The potential feasibility of an attenuated vaccine approach for HIV-1 was evaluated by challenging previously HIV-1 infected chimpanzees with a different strain of virus and monitoring whether "superchallenge resistance" occurred thereby preventing establishment of a second virus infection. Two chimpanzees, previously exposed to HIV-1IIIB 3 and 7 years ago, have been inoculated with escalating doses of HIV-1DH12 since September 1993. In May 1993, for example, one animal received 10,000 TCID50 units of virus and the other 10 ml of blood from a previously HIV-1DH12 infected chimpanzee. Both animals remain protected from infection by HIV-1DH12 as monitored by virus isolation and PCR. Mutations affecting both conserved and variable domains of gp120 have been studied. An amino acid substitution that eliminated a highly conserved glycosylation site in the C2 (second conserved domain) region of gp120 had no effect on synthesis, processing or release of gp120 but drastically reduced virus infectivity. Long-term propagation of this C2 mutant gave rise to a "second-site" (in C1 or V3) revertant viruses that exhibited wild type infection kinetics. Biochemical analyses or the C2 mutant, the revertant, and wild type virus particles revealed that the mutant particles contained no gp120; this "association" defect, affecting the non-covalent linkage between gp120 and gp41, was restored by the revertant changes. This result suggested that the reciprocal interaction of C1, C2 and V3 are critical for the retention of gp120 on progeny virions. The unique properties of V3 domains derived from macrophage tropic HIV-1 isolates (conferring tropism for monocyte derived macrophages [MDM], PBLs but not for T cell lines) were studied by replacing this region of a prototype T-cell tropic cloned provirus (NL4-3) with several V3 segments from MDM-tropic HIVs. The resultant viruses lost the capacity to infect T-cells and the virion-associated envelope protein acquired altered physical and functional properties: marked reduction in the spontaneous release of gp120; lower amounts of virion associated gp120 (per particle) than the parental HIV NL4-3; and impaired binding of virions to CD4.